Illuminating biology at the nanoscale with single-molecule and super-resolution imaging.

Speaker: Ziaowei Zhuang
Department: Chemistry (Harvard)
Location: TU Delft
Date: January 12, 2017
Author: Teun Huijben

Ziaowei Zhuang is professor of Chemistry, Chemical Biology and Physics at Harvard University. She gives her talk at the Dies Natalis of the TU Delft after receiving an honorary doctorate. Ziaowei Zhuang is introduced by Chirlmin Joo who tells us that Ziaowei is a remarkable researcher, especially because she invented two very important new techniques nowadays widely used in research, namely FRET and STORM. Keeping this in mind, I am curious what she will tell about it.

Ziaowei begins her talk by emphasizing why imaging and in particular molecular imaging is very important for both medical care as fundamental research. She reminds us of the fact that normal light microscopy is limited by the diffraction limit and that conventional microscopes cannot image details smaller than half the wavelength of light (~200 nm). To solve this problem she and her lab invented the STORM technique (stochastic optical reconstruction microscopy) approximately 10 years ago. STORM is a new type of fluorescent microscopy in which the fluorescent dyes are not constantly emitting light, but do this in a stochastic way by going on and off. If two points are very close to each other, a conventional microscope would give an image where the points are depicted as one, because the point spread functions of the two light sources are so close to each other, that they cannot be determined separately. However, with STORM the two points will emit light one by one and not at the same time. When the centers of the two point spread functions are determined, they will have different locations and the points are seen as two different sources (figure 1).

storm_principlefigure 1: Principle of STORM. In conventional microscopy all fluorphores are visualized at the same time, resulting in a blurry image when the light sources are close to each other. In STORM the fluorophores are stochastically activated, whereafter their centroid is calculated and the image rendered. In this way a higher resolution is obtained, since two points close to each other can be distinguished.[1]

They further improved this technique by using a cylindrical lens, which makes the point spread function asymmetrical depending on the z-position (figure 2). This enables 3D imaging with resolutions of ~20 nm in the x- and y-direction and ~50 nm in the z-direction, which is ten times better than the conventional microscope.

cylindrical_psf

figure 2: Cylindrical lens to image in 3D. When a cylindrical lens is used, it distorts the shape of the point spread function into an ellipse which shape is dependent on the z-position. Calculating this shape allows to determine the z-position of the molecule. 

The second part of her talk Ziaowei explains the concept of transcriptome imaging. The transcriptome is the collection of all the RNAs in a cell. If it were possible to image this transcriptome in some way, the sub-cellular organization of the RNA inside one cell or the spatial organization of transcriptomes in tissues can be visualized. In this way it would be possible to distinguish different functional cells in the same tissue by their transcriptomes.

The method is based on the single-molecule FISH technique in which small fluorescently labelled single-stranded pieces of DNA hybridize with the host DNA and give different chromosomes a different color. However, in transcriptome imaging we talk about thousands of different RNAs so a different probe is needed for each unique RNA. This is impossible, because it would be an immense task to make them and it is not possible to distinguish this amount of different color in the given visible light spectrum. To solve this, Ziaowei and her group came up with the brilliant idea of making only 16 different small constructs and turn them on sequentially (explained in figures 3 and 4). In this way the RNA distribution in a single cell or tissue can be visualized.

transcriptome_imaging
figure 3: Principle of transcriptome imaging. Each round only one of the 16 constructs is activated resulting in a fluorescent signal which is recorded. Only the RNAs containing this sequence will emit a signal. After repeating this procedure N-times many different RNA species can be identified by their unique sequence of emitted signals (figure 4). [3]  

transcriptome_imaging2
figure 4: RNA determination in transcriptome imaging. (B) All the results of 16 rounds of imaging, different colors mean different constructs and each dot indicates a potential RNA molecule. (C) Images after every hybridization step of the inset of figure B. White circles indicate possible RNAs. When one position gives a singel in multiple rounds, the sequence of signals on this position enables determination of the RNA species. 

After all I found the talk of Ziaowei very interesting. Especially knowing that she invented two widely used biophysical techniques FRET and STORM, which are both covered in the Nanobiology curriculum. Given that she is busy developing this new promising transcriptome imaging technique makes her even more special.

[1]: Single-molecule localization microscopy:

https://cam.facilities.northwestern.edu/588-2/single-molecule-localization-microscopy/

[2]: 3D STORM:
http://bme240.eng.uci.edu/students/08s/mkotlarc/developments.html

[3] Kok Hao Chen et al. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348 (2015).

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